This invention relates to nucleic acid and amino acid sequences of a delayed rectifier potassium channel subunit and to the use of these sequences in the diagnosis, treatment, and prevention of cancer, cardiovascular disorders, and neuronal disorders.
Ion channels are integral membrane proteins, typically comprised of four subunits, that form highly selective and tightly regulated pores in cellular membranes. Each of these pores controls the influx and efflux of a given ion (e.g., sodium, potassium, calcium, or chloride) across the plasma membrane or the membranes of intracellular compartments. Essential physiological processes, such as synaptic transmission, stimulation of secretion, fertilization, muscle contraction, and regulation of ion concentrations and pH, depend on the control of ion gradients by ion channels.
Ion channels open in response to various stimuli. For example, there are ligand gated channels, second-messenger gated channels, voltage gated channels, and shear- or stress-gated channels, as well as leak channels through which ions flow without specific stimulus. The gating properties characteristic of a given channel include the period of time it is open (open time), frequency of opening, strength of stimulus required for activation, and the refractory period. These characteristics can vary widely based on the subunit composition of the channel, association with accessory proteins, phosphorylation state, and post-translational modification.
Potassium channels are located in all cell types. In neurons and other excitable cells, they set resting membrane potential, regulate key aspects of the action potential including duration, frequency, and pattern of discharge, and are responsible for repolarization following an action potential. In non-excitable tissue, potassium channels are involved in essential physiological processes including cell protein synthesis, control of endocrine secretions, and the maintenance of osmotic equilibrium across cell membranes and in the plasma. Categories of potassium channels include voltage-gated potassium channels, ATP-sensitive potassium channels, second messenger-gated potassium channels, and calcium-activated potassium channels.
Like the voltage-gated channels for sodium. and calcium, voltage-gated potassium channels (VGKC) are composed of four polypeptides that form homo-oligomers or hetero-oligomers to create the pore through which potassium ions flow. At least ten of these potassium-pore-forming subunits, or xcex1 subunits, have been described that fall into four families, designated Kv1-Kv4. Examples of a subunits include the HERO (human ether a go-go) subunit, named after a Drosophila homolog, and the KV(LQT)1 subunit. These xcex1 subunits share a common structural organization which is similar to the xcex1 subunits of other voltage-gated channels. There are six transmembrane-spanning domains with a short region between the fifth and sixth transmembrane regions that senses membrane potential, and the amino and carboxy termini are located intracellularly. Current flow through a VGKC can be either an A-type current, which activates at sub-threshold membrane potentials and rapidly inactivates, or a rectifier type current, which activates and inactivates slowly.
The potassium channel xcex2 subunit, also known as the minK protein or the cardiac delayed rectifier potassium channel protein, is an accessory subunit that regulates potassium channel gating characteristics, but does not participate in formation of the potassium pore. At least four variants of this 129 amino acid peptide have been described. They contain a single transmembrane domain, and have been shown to assemble with at least two different VGKC pore-forming subunits and regulate their gating characteristics. For example, in mammalian heart, the duration of ventricular action potential is controlled by an inward, rectifying, delayed-type potassium current, which has fast- and slow-activating components. Two xcex1 subunits have been shown to mediate this complex current: the HERG subunit, and the Kv(LQT)1 subunit. The fast-activating potassium current is mediated by a complex of minK and HERG, while the slow-activating component is mediated by minK and Kv(LQT)1. Thus, minK is central to the control of heart rate and rhythm. (Folander, K. et al. (1994) GI 452494; Lai, L. P. et al. (1994) Gene 151:339-340; Sanguinetti, M. C. et al. (1996) Nature 384:80-83;McDonald, T. V. et al. (1997) Nature 388:289-292.)
Potassium channel dysfunctions are associated with a number of disease states. For example, potassium channels in smooth muscle tissue of the circulatory system are implicated in hypertension, while those channels in the kidney are involved in hypokalemia and the associated Bartter""s syndrome and Getelman""s syndrome. Both of these syndromes are characterized by alterations in potassium metabolism in the kidney. Potassium channels are also involved in certain neuronal disorders. Epileptic seizures can be induced by agents (e.g., pentylenetetrazol) which block potassium channels, most likely due to the loss of regulation of cellular membrane potentials. A potential role for potassium channels in Alzheimer""s disease has been suggested by studies demonstrating that a significant component of senile plaques, beta amyloid or A beta, also blocks voltage-gated potassium channels in hippocampal neurons. (Antes, L. M. et al. (1998) Seminar Nephrol 18:31-45; Stoffel, M. and Jan, L. Y. (1998) Nat. Genet. 18:6-8; Madeja, M. et al. (1997) Eur. J. Neurosci. 9:390-395; and Good, T. A. et al. (1996) Biophys. J. 70:296-304.)
Altered function of minK proteins is involved in long QT syndrome, a cardiovascular disorder in which patients experience cardiac arrhythmias, fibrillation, syncope and sudden death. Another cardiovascular disorder, Jervell and Lange-Nielsen syndrome, is also produced by mutations in minK. Jervell and Lange-Nielsen syndrome is characterized by abnormal ventricular repolarization, syncope and sudden death, and is also associated with congenital deafness. In both instances, mutations in the gene encoding a minK homolog are responsible for altered gating characteristics of the VGKC, which results in dysfunctional potassium channels. (Schulze-Bahr, E. et al. (1997) Nature Genet. 17:267-268; and Splawski, I. et al. (1997) Nature Genet. 17:338-340.)
The discovery of a new delayed rectifier potassium channel subunit and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of cancer, cardiovascular disorders, and neuronal disorders.
The invention is based on the discovery of a new human delayed rectifier potassium channel subunit (DRPCS), the polynucleotides encoding DRPCS, and the use of these compositions for the diagnosis, treatment, or prevention of cancer, cardiovascular disorders, and neuronal disorders.
The invention features a substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides a substantially purified variant having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as an isolated and purified polynucleotide which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides an isolated and purified polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2, and an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention also provides an isolated and purified polynucleotide having a sequence complementary to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2.
The invention further provides an expression vector comprising at least a fragment of the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, the method comprising the steps of: (a) culturing the host cell comprising an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as a purified agonist and a purified antagonist of the polypeptide.
The invention also provides a method for treating or preventing a cancer, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for treating or preventing a cardiovascular disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising substantially purified polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for treating or preventing a neuronal disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for detecting a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO: 1 in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in the biological sample. In one aspect, the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.